The presence of clonally expanded populations of T cells and B cells in the inflamed tissue of patients with type 1 diabetes (diabetes) and in multiple sclerosis (MS) has long suggested that self-antigens are driving the disease process. This PPG had brought together investigators applying highly sensitive, novel techniques including the use of self-antigen tetramers and single cell cloning methods to interrogate the antibodies and T cells in the pancreatic lymph nodes of patients with diabetes and brain tissue of patients with MS. We have made significant progress in providing new evidence for the role of two candidate antigens, insulin and MOG, in these disease processes. These findings, along with the recent surprising results that infusions of anti-CD20 mAb therapy targeting B cells may have efficacy in human autoimmune diseases has led us to the underlying hypothesis of this PPG effort that alterations in the function of autoantigen-specific B cells, acting as antigen presenting cells, are central in the induction of human autoimmune diseases. In Aim 1, we will complete our initial efforts to identify the autoantigens driving clonal expansion of B cells in MS plaques and now examine diabetes islet tissue and lymph nodes. We will determine BCR sequences of individual B cells isolated by laser microdissection, followed by affinity purification of antigens with reconstructed antibodies and molecular definition of targets by tandem mass spectrometry. In Aim 2, we will interrogate the autoreactive B cells by a novel, nanowell-based technology and determine whether the B cells of patients with autoimmune diseases are pro-inflammatory. This interrogation of defined antigen specific cells will focus on functional cytokine secretion and phenotypic cell surface markers. In Aim 3, we will examine whether B cells and autoantibodies present at the sites of inflammation directly contribute to disease pathology through autoantigen presentation. In collaboration with the Kuchroo lab, we will study the ability of human recombinant antibodies generated in Aim 1 to induce demyelination in EAE models as well as the ability of B cells that express human BCRs to induce differentiation of autoreactive T cells into pathogenic effectors. High-affinity insulin-specific autoantibodies recognize the same insulin A1-15 chain epitope as human T cells, and we will examine whether these BCRs protect the T cell epitope from premature degradation during antigen presentation, a question that is clinically relevant because the affinity of insulin autoantibodies correlates with progression to diabetes. The clear association of autoantibodies with predicting susceptibility to autoimmune diabetes and the presence of anti- MOG autoantibodies in MS plaque tissue together suggest the importance of investigating B cells in these human autoimmune diseases.